The gastrointestinal (GI) system has a well-organized developmental architecture which includes intestinal stem cells (ISCs), transient amplifying (TA) progenitors, functionally mature cells, and apoptotic cells all of which are confined to identifiable regions in each crypt/villus unit. This developmental architecture forms a sequential array of compartments (or zones) which promote self-renewal of stem cells, proliferation of progenitors, differentiation of progenitors to mature cells, and apoptosis in the mature cells. The developmental architecture or microenvironment is generally divided into three functional compartments, based upon stages of stem cell development, including (1) self-renewal, (2) expansion or transient amplification, and (3) differentiation zones. These zones correspond to the developmental state of the ISCs.
The mucosa of the small intestine is involved in nutrient absorption and is characterized by evaginations into the villi, and at the bases of the villi, by short tubular invaginations into crypts. The villi are projections into the lumen and are covered predominantly with mature, absorptive enterocytes, along with occasional mucous-secreting goblet cells. At the base of the crypts are the ISCs, which continually divide and provide the source for all epithelial cells in the crypts and villi. ISCs are multipotent, undifferentiated cells that fundamentally retain the capacity for cell division and regeneration to replace various intestine cells that undergo apoptosis and die. They are thought to be located in the fourth or fifth position from the bottom of each crypt in the small intestine. ISCs are also found at the bottom of the table region of the villi of the large intestine. Recently crypt base columnar (CBC) located at the 0 position was shown to have stem cell properties. Unlike adult stem cells in other tissue systems, and for an unknown reason, the currently identified ISCs have a relatively high rate of cell proliferation. Consequently, ISCs provide a general system for studying stem cells and the regulatory mechanisms that govern their proliferation, growth, and differentiation. In particular, this system is suitable for examining tumor development and recurrence, as well as, evaluating cancer therapies.
It has been proposed that the failure of most current therapies to completely cure cancer is caused by “cancer stem cells,” which are resistant to therapy aimed at destroying malignant proliferation. Recently, several lines of evidence demonstrated the existence of tumor-initiating cells in acute myeloid leukemia, breast cancer, and brain cancer. In these studies, cancer stem cells could be recognized by their ability to initiate tumors upon transplantation into a secondary host. Still, in vivo evidence, directly linking the mutated stem cells with initiation of neoplasms in the natural setting, is lacking. Further, these putative “cancer stem cells” have yet to be identified.
While there are many “causes” of intestinal cancer, it is well established that almost all cases begin with the development of benign polyps in the epithelial layer of the intestine. The epithelium of the small intestine is composed of a proliferation compartment (crypt) and a differentiation compartment in the villus region (FIG. 1A). Intestinal stem cells (ISCs), located at the 0, 4 or 5th position from the crypt bottom and above Paneth cells, are the driving force for intestinal regeneration. ISCs give rise to daughter cells that undergo further proliferation in a transient-amplifying compartment (TAC), producing absorptive enterocytes and secretory lineages including goblet and enteroendocrine cells, as well as Paneth cells which are located at the crypt bottom. Several signal pathways are known to be involved in regulating this complex sequence of events. However, the molecular mechanisms controlling ISC numbers and the relationships between the signaling pathways that govern intestinal homeostasis are not completely understood.
Intestinal polyposis (IP) features a substantial increase in the number of crypts and a reduction in epithelial cell differentiation. A still unanswered question for IP, and for neoplasia in general, is whether stem cells are involved in polyp initiation. An increase in stem cell number has been proposed to be responsible for crypt expansion, but direct in vivo evidence for this proposal is still lacking.
Studies of human hereditary IP syndromes and equivalent animal models, which typically but not uniformly predispose affected individuals to GI cancers, have provided substantial insight into the genetic control of intestinal homeostasis, polyposis, and cancer. Polyposis can result from impaired bone morphogenetic protein (BMP) signaling, or by over-activation of Wnt/β-catenin signaling. Wnt/β-catenin signaling exert positive control on multiplication of both stem cells and crypts; whereas, BMP signaling restricts stem cell number and prevent polyposis in part by suppressing Wnt signaling.
Cowden disease (CD) (OMIM #158350) is a rare autosomal dominant disorder featuring multiple hamartomatous lesions including intestinal polyps. CD results from mutation in the tumor suppressor gene PTEN, which encodes a lipid and protein phosphatase that acts as a negative regulator of the phosphatidylinositol-3-OH kinase (PI(3)K)-Akt pathway. The roles of PTEN and Akt in intestinal homeostasis and polyposis have not been defined, but there is evidence that they could be key players in the interplay between Bmp and Wnt signals. Mutation of BMP receptor 1A can result in a Cowden-like syndrome resembling loss of PTEN. BMP signaling may in part be mediated by inhibition of PI3K/Akt activity via positive regulation of PTEN. In turn, PTEN inhibits while Akt enhances the nuclear localization of β-catenin. Likewise, the PI3K inhibitor Ly294002 can block nuclear localization of β-catenin and its transcriptional activity. It has been proposed that Akt assists Wnt signaling in promoting activation of β-catenin in ISCs. Akt can regulate β-catenin activity indirectly, through GSK3β, or directly, through phosphorylation of β-catenin which enhances β-catenin's nuclear function.
It is desired to develop compositions and methods for the induction of ISC self-renewal, proliferation, growth, and differentiation within the intestinal tissue architectural structure. Methods for controlling the molecular mechanisms that, in turn, control intestinal pathways are also desired. Identification of cell markers, including cell surface markers, that are useful for ascertaining the role of stem cells in crypt expansion are desired. It is especially desired to identify distinct markers, which can be used to identify various types of cells such as “cancer stem cells” in the tissue.